This proposal is aimed at defining the apoptosis signaling mechanisms mediated by cyclooxygenase-2 (COX-2) inhibitors in prostate cancer cells, and represents part of our effort to develop a new class of therapeutic agents against hormone-refractory prostate. cancer (HRPC). The hypothesis to be tested is twofold. First, we propose that the mechanism underlying COX-2 inhibitor-induced apoptosis is independent of COX-2 inhibition. Second, we propose that sulfonamide. COX-2 inhibitors (e.g., celecoxib) induce apoptosis in prostate cancer cells by interfering with multiple signaling components. Putative targets include protein phosphatase and sarco-endoplasmic reticulum Ca2+- ATPase (SERCA) pumps. Interactions with these targets lead to concurrent down-regulation of Akt and ERK phosphorylation and cytosolic Ca2+ perturbation, respectively, resulting in rapid apoptotic death. These hypotheses will be tested using celecoxib and its structural analogues in a panel of prostate carcinoma cells that display different androgen responsiveness and genetic lesions. Specific aim 1 is to verify the hypothesis that the induction of apoptosis is independent of COX-2 inhibition. The expression levels of COX-2 in prostate cancer cells will be down-regulated by antisense COX-2 cDNA, and the consequent effect on cell viability and on sensitivity to COX-2-inhibitor-induced apoptosis will be examined. Specific aim 2 is to identify the molecular target through which celecoxib and its analogues exert the down-regulation of Akt and ERK phosphorylation. An integrated approach is undertaken to examine the effect of celecoxib, both in vitro and in vivo, on protein phosphatases and upstream kinases responsible for Akt and ERK phosphorylation. In addition, photoaffinity analogues of celecoxib will be prepared to aid the identification. Specific aim 3 is to investigate the role of Ca2+ in celecoxib-induced apoptosis in prostate cancer cells. Our data showed that celecoxib is an inhibitor of SERCA pumps, reminiscent of thapsigargin. Thus, it represents a unique pharmacological tool to study the effect of cytosolic Ca2+ increase on apoptosis signaling. We propose a Ca2+ clamping experiment to investigate whether celecoxib- induced Ca2+ perturbation is required for Akt and ERK down-regulation and the consequent rapid induction of apoptosis. Information obtained from these studies will have profound translational potential for new drug development for HRPC. Identification of new anti-cancer agents whose mode of action is distinct from existing chemotherapeutic regimens will foster new therapeutic strategies.